PROJECT SUMMARY/ABSTRACT (DESCRIPTION) Advances in DNA sequencing technologies have uncovered remarkable structural complexities within the human cancer genome, including a new category of massive and localized intra-chromosomal rearrangements coined chromothripsis. Since its discovery in 2011, the signatures of chromothripsis have now been detected in a broad spectrum of solid and hematological tumors. These alterations are thought to occur during a single catastrophic event; however, their underlying mechanistic origins are not well understood. Errors in mitotic cell division can provoke chromothripsis through entrapment of missegregated chromosomes into aberrant structures called micronuclei. Dr. Ly previously identified that chromosomes in micronuclei are subjected to extensive shattering in mitosis, and the resulting DNA fragments are reassembled by canonical end-joining repair in the subsequent interphase. This proposal for an NIH Pathway to Independence Award seeks to understand how chromosome segregation errors during mitosis initiate a cascade of downstream genomic instability events to directly shape or contribute to the cancer genome. In the first aim, examples of fully functional chromosomes with de novo chromothripsis will be generated through development of a powerful and reversible centromere-specific inactivation approach coupled to a chromosome-specific selection strategy in human tissue culture cells. Genome-wide sequencing will be used to identify the hallmark features of chromothripsis from unique clonal derivatives following chromosome missegregation into micronuclei. In the second aim, the precise mechanisms and spatiotemporal dynamics of chromosome shattering and reassembly events will be explored through multidisciplinary cell biological approaches, including gene disruption and live- cell imaging. The third aim will focus on how chromosomes with gross rearrangements that lack a functional alphoid centromere are able to propagate indefinitely through the epigenetic formation of a stable, new centromere at non-alphoid loci. By leveraging his expertise in engineering sophisticated tissue culture models combined with his background in cancer biology, these collective efforts by Dr. Ly will establish the mechanisms and consequences of mitotic errors in triggering genomic instability ? insights that are critical for understanding the biogenesis of complex genomic features commonly manifested in patient tumors. During the mentored K99 phase of the Award, Dr. Ly will receive additional and needed training at the Ludwig Institute for Cancer Research under the guidance of Dr. Don Cleveland ? a widely recognized and established leader in the fields of cell division and aneuploidy. The Ludwig Institute for Cancer Research, the University of California at San Diego, and the surrounding La Jolla scientific community serves as an exceptional atmosphere for research training, collaborative science, and career development. An excellent team of collaborators (Drs. David Page and Andrew Shiau) and consultants (Drs. Richard Kolodner, Paul Mischel, and Karen Oegema) has been assembled to provide Dr. Ly with additional scientific training and career support before, during, and after transitioning toward an independent tenure-track faculty position. The R00 Award phase will set the foundation for Dr. Ly's long-term career goals of establishing a rigorous research program that seeks to address exciting, unanswered questions in the fields of cell biology, cancer genetics, and genome stability through use of cutting-edge, interdisciplinary methodologies.